The principal aim of the proposed research is to elucidate the molecular basis of visual excitation in mammalian retinal rod cells. We want to define the molecular and conformational links between the cis-trans isomerization of rhodopsin in the disc membrane and the change in sodium permeability of the plasma membrane. Several major lines of research are in progress in this laboratory: (1) We have found that several proteolytic enzymes cleave rhodopsin in retinal disc membranes into large fragments, F1 and F2, which form a non-covalent complex that is red and regenerable after bleaching. Most interestingly, light dissociates this complex. We are using chemical and spectroscopic techniques to investigate the hypothesis that the photodissociation of F1 and F2 is an expression of a critical conformational change in visual excitation. (2) We are mapping the structure of rhodopsin by energy transfer spectroscopy. Specific sites on rhodopsin are being labeled by fluorescent probes. For example, we have used transglutaminase to label the junction of the F1 and F2 fragments. The distances between these sites are determined by energy transfer measurements. (3) We have devised a new rapid-flow technique for obtaining the resonance Raman spectra of rhodopsin and other photolabile molecules. We are obtaining the resonance Raman spectra of model retinal derivatives and of photolytic intermediates to delineate the conformational changes in retinal following the absorption of a photon. (4) We have carried out neutron diffraction studies of intact retinas at Brookhaven National Laboratory and have obtained a low-resolution Fourier synthesis. We plan to determine the location of the retinal chromophore by the difference Fourier method, using retinas containing deuterated retinal.